CZ307026B6 - A method of preparing a surface of a substrate comprising carboxybetaine functional groups - Google Patents

Abstract

The present invention relates to a method of preparation of a substrate surface containing carboxybetaine groups with bound bioactive substances, having resistance to undesirable deposition of biological media components on the substrate surface, comprising the steps of chemical activation of carboxybetaine groups on the surface of the substrate by converting the carboxybetaine carboxyl group into an active ester, covalent binding of a bioactive substance to some of the active esters, and reaction of the product from the previous step with an acid of general formula (I) NH2-(CH2-CH2-0)n-CH2-COOH, where n = 1 to 4, wherein the acid of general formula (I) reacts with those active esters, which have not undergone covalent bonding of the bioactive substance.

Description

Technical field

The invention relates to a process for preparing a surface of a substrate containing carboxybetaine functional groups with bound bioactive substances, which increases the surface resistance to undesirable biological deposition upon contact with biological media.

BACKGROUND OF THE INVENTION

When virtually all common materials come into contact with a biological medium, a biological fouling occurs on their surface, beginning with the adsorption of biological molecules, mainly proteins, to which cells and microorganisms can adhere, depending on the composition of the medium, followed by other biological processes such as blood coagulation. inflammatory and immune responses or bacterial biofilm formation. The resulting biological deposits may impair the function of materials and devices that operate in biological media, such as body fluids, media containing cells, food and media from biological productions, and the biological environment in general. The problem is particularly critical for materials used in contact with blood serum, plasma or blood. Therefore, surfaces that prevent the non-specific formation of biological deposits in biological media while allowing the binding of bioactive elements mediating specific surface interaction with target components of the biological environment are very important for many biotechnological and medical applications, such as biosensors, membranes and particles for separation and accumulation biological agents and cells, drug carriers and diagnostic particles applied to the bloodstream, materials in contact with blood, and cell carriers, scaffolds, for tissue engineering.

At present, surfaces prepared by grafting hydrophilic electroneutral polymers such as non-ionic poly (oligo (hydroxyethylene glycol) methacrylate)) (polyHOEGMA), poly (2-hydroxyethyl methacrylate) (polyHEMA) are considered to be most resistant to biological deposition (antifouling). , poly (3-hydroxypropyl methacrylate) (polyHPMA), poly (A- (2-hydroxypropyl) methacrylamide) (polyHPMAA) and zwitterionic poly (carboxybetain methacrylate) (polyCBMA) and poly (carboxybetain acrylamide) (polyCBAA), from substrate surfaces ( by surface-initiated atomic transfer polymerization (SI ATRP). The resulting polymer brush is a layer of densely arranged polymer chains bonded to the surface at one end. PolyCBAA, polyCBMA and polyHPMAA brushes are the only brushes that effectively suppress the deposition of undiluted blood plasma and serum.

An alternative method is to graft it to polymer chains prepared by solution polymerization. The lower density of polymer chains achieved in brushes prepared by this method compared to brushes prepared by the "grafting from" method provides the surface with less resistance to biological deposition.

Even weaker resistance especially to plasma and serum deposition is provided by frequently used carboxymethyldextran layers or coatings of self-organized monolayers (SAMs) from a mixture of molecules (CH 2 O 3 O 2 (CH 2) n (EO) ₆ OOH, where EO is ethylene oxide. oligoethylene oxides containing, in some positions, carboxyl groups used to bind bioactive substances.

Activation of the hydroxyl groups in their side chains is used to bind bioactive substances to non-ionic polymer chains. The residual products of this activation, which remain on the polymers after the binding of the bioactive substances, greatly impair the resistance of the brushes to biological deposition. Surfaces with the desired biological activity and better resistance to deposition were prepared by binding bioactive substances to polyCBMA or polyCBAA brushes having carboxybetaine zwitterions as side groups of polymer chains. Card

PolyCBMA and polyCBAA with bioactive substances attached to carboxybetain side groups of polymers are included, inter alia, in US 2014/0 370 567 and US 2013/0 244 249. Patent application PV 2015-313 discloses poly ( HPMAA-coCBMAA) grafted by copolymerization of HPMAA monomers and carboxybetainacrylamide (CBMAA) from the surface of various substrates including polymer nanoparticles and binding of the bioactive agents to their activated carboxybetain groups. WO 2016/177 354, which follows PV 2015-313, also includes the preparation of poly (HPMAA-co-CBMAA) brushes prepared by solution polymerization and covalently grafted to the substrate surface. BACKGROUND OF THE INVENTION Biodegradable poly (HEMA-co-CBMAA) copolymers (Kostina, et al., Biomacromolecules 2012, 13, 4164—4170) or sorbents whose surface is modified by the attachment of carboxybetain zwitterions (WO 2014 / 165,421 A1).

Bioactive substances containing one or more amino groups are bound to the carboxy betaine groups practically exclusively by reaction with the carboxy betaine zwitterionic carboxyl which is first activated to an intermediate readily reacting with the nucleophilic amino group of the linked bioactive substance. This activation is mainly carried out by reaction with various N-substituted carbodiimides (DCI) to form the active ester of O-acylisourea. However, this intermediate has an extremely low lifetime in the aqueous environment and is rapidly hydrolyzed, so that the amino-coupling reaction has an insufficient yield. For this reason, the reaction is carried out in the presence of N-hydroxysuccinimide (NHS) or its derivatives, which reacts rapidly with O-acylisourea to form the active NHS-ester. The NHS ester has a higher reactivity to the amino group than O-acylisourea and is more stable to hydrolysis, so that the desired yield is better achieved. Reaction of both the NHS-active ester and the O-acylisourea with an amino group leads in both cases to the same binding of the bioactive agent to the carboxybetain group by an amide bond.

Unreacted NHS-active esters formed by activation of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS) of only carboxyl groups (not carboxybetaines), eg on the surface of the above SAMs, self-hydrolyze back to carboxyl groups. Deactivation of NHS-active esters improves surface resistance to biological deposition, therefore, incubation with ethanolamine solution (US 5,561,069 A) is often used to better inactivate the active esters remaining after the binding of bioactive substances to carboxyl groups in SAMs or carboxymethyldextran. Deactivation of aldehyde groups on modified polyethylene glycol, PEG-CH = O, by reaction with ethanolamine and glycine has also been reported (Wildling et al. Bioconjugate Chem. 2011, 22, 1239-1248), however, glycine deactivation resulted in an increase in non-specific deposition compared to ethanolamine. , possibly due to the introduction of excess carboxyl charge.

Although it was generally believed that both the O-acylisourea intermediates and the NHS-active esters that did not react with the amino group can be quantitatively hydrolyzed back to the carboxyl groups after the coupling reaction, the carboxyl groups of the carboxybetain zwitterions are not regenerated by DCI / NHS activation. Our studies based on reflective IR spectroscopy have shown that there is no quantitative regeneration of carboxyl groups when carboxybetaines are reacted with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), which is the most commonly used DCI, and NHS does not or by prolonged exposure to aqueous solutions with different electrolyte compositions, ionic strength and pH.

Given the extent and breadth of use of carboxybetaine groups to bind biologically active substances to various surfaces, the regeneration of unreacted active esters to a carboxy-terminated molecule appears to be key to reducing the fouling of these surfaces.

US 7 943 370 B2 describes the preparation of the surface of a substrate containing carboxybetaine groups by converting the carboxybetaine to the active ester, covalently binding the bioactive agent and deactivating unreacted active esters with glycine or β-alanine. Documents Chou Y.-N. et al., Acta Biomaterialia 2016, 40, 31-37 and Vaisocherová H. et al., Biosensors and Bioelectro

-2E 307026 B6 nics 2014, 51, 150-157 discloses the preparation of the surface of a substrate containing carboxybetaine groups by converting the carboxybetaine to the active ester, covalently binding the bioactive agent and deactivating unreacted active esters with glycine. WO 2009/130 233 A1 describes the preparation of halogenated surfaces with a potential content of carboxybetaine groups by converting the carboxyl group into an active ester, covalently binding the bioactive agent and deactivating unreacted active esters with glycine. Vaisocher, H. et al., Biosensors and Bioelectronics 2014, 51, 150-157 describe the preparation of a substrate containing carboxybetaine groups by converting the carboxybetaine to the active ester, covalently binding the bioactive agent and deactivating unreacted active esters with glycine.

SUMMARY OF THE INVENTION

The present invention relates to the regeneration of active carboxybetain group esters by a method of preparing a substrate surface containing carboxybetain functional groups which results in the replacement of unreacted active esters remaining after binding of the bioactive agent to some of the activated carboxybetain zwitterions with (2-aminoethoxy) acetic acid. Parts of a product or device whose surface is to perform an active function in a biological medium that could be affected by biological deposition, and whose surface contains carboxybetaine groups, may be of organic or inorganic materials, may have any morphology, eg particles, membranes, tubes, hoses, wafers, porous material, fiber networks, and various uses in contact with biological media, such as biosensors, affinity particles and membranes for separation and accumulation of biologicals, targeted drug delivery carriers, tissue engineering biomaterials, antithrombogenic materials for blood contact.

The present invention consists in immediately after the activation of the carboxybetaine groups and the binding of biologically active substances, the substrate surface is incubated with a (2-aminoethoxy) acetic acid solution. During this incubation, the terminal amino group of this molecule reacts with active esters not consumed upon prior binding of the biologically active substances. The free carboxyl group in the portion of the molecule that replaced the active ester forms a new zwitterion with a quaternary nitrogen cation remaining after activation of the original carboxyl group. This procedure neutralizes the reactivity of residual active esters and at the same time compensates for the positive charge of the quaternary nitrogen cations, thus again increasing the surface resistance to biological deposition. The technical effect of the process of preparing the surface of a substrate containing carboxybetaine functional groups is particularly (but not limited to) high-density polymeric brushes containing carboxybetaine side groups. Small activating agent molecules can penetrate between the polymer chain and react with carboxybetains throughout the brush volume, whereas only carboxybetains near the surface of the layer are available for binding large bioactive substances, eg proteins.

It is an object of the present invention to provide a surface preparation of a substrate containing carboxybetaine groups, with increased resistance to undesirable deposition of biological media components on the substrate surface, comprising the steps of:

(a) chemical activation of carboxybetain groups on the surface of the substrate by converting the carboxyl group of the carboxybetain to the active ester;

b) covalently attaching the bioactive agent by reacting its amino group with the active ester prepared in step a);

wherein after step b), step c) is carried out in which the product of step b) is reacted with (2-aminoethoxy) acetic acid,

Wherein the active esters are reacted with (2-aminoethoxy) acetic acid and the bioactive agent is not covalently bound in step b). Preferably, step c) is carried out at basic pH, more preferably at a pH in the range of 7.5 to 9, most preferably at pH 8.

The carboxybetaine groups may either be directly part of the substrate material or may be attached to the surface of the substrate, eg as part of molecules grafted to the surface of the substrate, or the substrate may be coated with such groups, eg polymer brush prepared by surface initiation or prepared by attaching polymer chains prepared by solution polymerization to a surface (grafting to).

Carboxybetaine groups are well known to the person skilled in the art and are generally defined as neutral chemical groups comprising a quaternary ammonium cation which carries no hydrogen atom and a negatively charged carboxyl group not adjacent to the quaternary ammonium cation (IUPAC, Compendium of Chemical Terminology).

For the purposes of the present invention, a substrate is an object that is resistant to the deposition of biological media components or that needs to be coated with a polymeric layer conferring it resistance to the deposition of biological media components (can be grafted onto a polymer brush or coated with a polymeric coating). Thus, the substrate may be:

(1) an object whose surface is coated with a grafting from polymer surface brushing by live radical polymerization, see PV 2015-313 and WO 2016/177 354, (2) an object whose surface is coated with a layer of polymers prepared in solution and subsequently attached to the surface by covalent bonding or physical adsorption (see graphing to), see WO 2016/177 354, (3) an object whose surface is covered by radical initiated polymerization generated by chemical or physical activation of the object surface, (4) (5) an object whose surface can be modified by binding a molecule containing carboxybetain zwitterions, (6) an object that contains carboxybetain zwitterions in its structure, eg a polymer gel, where some monomer units contain carboxybetain zwitterionty.

The polymer layers formed on objects (1), (2), (3) and (4) each comprise at least one homopolymer or copolymer containing carboxybetaine zwitterions in the side chains. In the layers on objects (2) and (4), polymers containing carboxybetain zwitterions may be mixed with hydrophilic polymers, preferably selected from polyHPMAA, polyHOEGMA, polyHEMA, and polyHPMA.

Preferably, the layers on objects (1), (2), (3) and (4) are formed from a homopolymer selected from the group consisting of polyCBMAA, polyCBMA and polyCBAA or a poly (A-co-B) copolymer wherein A is a monomer unit selected from the group consisting of HPMAA, HOEGMA, HEMA, HPMA and B is a monomer unit at a concentration of 1 to 99 mol. % selected from the group consisting of CBMAA, CBMA, CBAA.

The shape, dimensions, morphology and chemical nature of the substrate is not critical, such as flame or variously shaped objects, tubes, fibers, particles, membranes, microparticles, nanoparticles, porous materials, metals, silicon, silicate or aluminosilicate based materials (e.g. glass), polymers, inorganic materials, etc.

-4GB 307026 B6

For the purposes of the present invention, a biological medium is a fluid containing biological substances, ie, biomolecules and their associates (proteins, carbohydrates, polysaccharides lipids, nucleic acids, lipoproteins, glycoproteins, organelles, etc.), viruses, cells, microorganisms and fragments thereof. Thus, the biological medium is, for example, blood and other body fluids, blood plasma and serum, tissue extracts, cell lysates and suspensions, food extracts.

A bioactive (biologically active) substance in the present invention is a substance containing at least one NH ₂ group, selectively interacting with a target component of the biological medium. The bioactive agent may have affinity for the target component, typically the bioactive agent is a natural antibody, antigen, lectin, and cell receptor, and artificial analogues and recombinantly prepared portions thereof, and synthetic oligopeptide sequences, nucleic acids and portions thereof, and synthetic oligonucleotide sequences and aptamers. A bioactive agent can catalyze the chemical conversion of a target agent, eg, an enzyme, a coenzyme, and synthetic analogues thereof. Alternatively, the bioactive agent may elicit a biological response, such as an anticoagulant, e.g., heparin, proteins and oligopeptide sequences responsive to cellular integrins, growth factors, hormones, and drug and vitamin derivatives. The bioactive substances can also be, for example, nanoparticles functionalized by the NH ₂ group, in particular metal, polymer, silicone nanoparticles, metal oxide nanoparticles or magnetic core polymer nanoparticles.

The active ester of carboxybetaine is the product of the reaction of the carboxyl group of the carboxybetaine with N-substituted carbodiimides, i.e. O-acylurea, and / or the reaction product of the carboxy group of the carboxybetaine with N-substituted carbodiimides and N-hydroxysuccinimide (NHS) or derivatives thereof. ester or sulfo NHS ester.

In a preferred embodiment, step c) is carried out by first rinsing the product of step b) with the buffer used as solvent in step b), followed by rinsing with the buffer that will be used in the next step for incubation, preferably such a buffer buffer of pH 8, then the product is incubated with a solution of (2-aminoethoxy) acetic acid in buffer, followed by rinsing with the buffer previously used for incubation, followed by rinsing with the solution into which it is then placed for storage or water and dried.

In one embodiment, the carboxybetaine groups are contained in the polymer layer on the substrate surface as a polymer brush prepared by polymerizing or grafting polymers onto the substrate surface, or the polymer coating, preferably the layer has a thickness in the range of 5 nm to 5 µm.

In a preferred embodiment, the polymer layer containing carboxy betaine groups is a polymer brush of polyCBMAA, polyCBMA, polyCBAA or poly (HPMAA-co-CBMAA / x mol%), wherein x (molar CBMAA concentration) is in the range of 1 mol. % to 99 mol. %. Preferably, the layer has a thickness in the range of 5 nm to 5 µm.

In one embodiment, the substrate is selected from the group consisting of particles, porous membranes, scaffolds, and biosensors.

The particles are preferably of a material selected from the group consisting of gold, silver, magnetic materials, silicon, SiO ₂ , polymers, falling within the definition of substrate (1) to (6) and preferably have a diameter of 5 nm to 1 mm. Particles with bioactive agents bound to activated carboxybetain groups on the particle surface, including the inner surface of the porous particles, are useful as carriers for targeted therapy and in vivo diagnostics, biological substance separation and accumulation, and enzymatic catalysis in bioreactors.

Porous membranes are membranes designed for affinity separation of biological substances from biological media.

-5GB 307026 B6

Cell carriers (scaffolds) are well known to those skilled in the art, for example, for use in tissue engineering (see e.g.

http://wikiskripta.eu/index.php/Tk%C3%A1%C5%88ov%C3%A9_in%C5%BEen%C3%BDr stv% C3% AD).

In one embodiment, the substrate is a biosensor detection surface for direct detection or multi-stage detection of analytes in complex biological media, eg, by optical or mass biosensors. Preferably, the detection surface is coated with a polymeric brush of polyCBMAA, polyCBMA, polyCBAA or poly (HPMAA-co-CBMAA) with bound bioreceptors. Bioreceptors in this application are bioactive agents having selective affinity for the target components of the medium to be analyzed.

In one embodiment, the substrate is for contact with blood in vitro, ex vivo, and in vivo.

The method of preparing the surface of a substrate containing carboxybetaine groups according to the present invention, which has increased resistance to undesired deposition of biological media components on the substrate surface, provides a very fast, cheap and efficient alleviation of undesirable biological media deposition problems over prior art solutions.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: General method of surface treatment of a substrate containing carboxybetaine functional groups

In step (a), the carboxybetaine groups present are chemically activated by converting the carboxybetaine carboxyl groups to the active NHS ester or sulfo-NHS ester by reaction with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and A -hydroxysuccinimide (NHS) or sulfo-NHS. In step (b), the sample is incubated with a solution containing bioactive substances (BALs) during which these substances are covalently bound to some of the active esters formed in step (a). In step (c), the active NHS esters or sulfoNHS esters that have not reacted with the bioactive agent are reacted with (2-aminoethoxy) acetic acid molecules. During incubation of the sample with an aqueous solution of (2-aminoethoxy) acetic acid, these molecules are covalently bound to the surface by a permanent amide bond.

Example 2: Resistance of carboxybetain polymer brushes to biological deposition from undiluted blood plasma and food samples after EDC / NHS activation, and after reaction with deactivating agents

The gold surface of the surface plasmon resonance (SPR) chip, a glass plate with a 50 nm thick steamed gold layer, was coated with a polyCBMAA brush or a copolymer brush containing 15 moles by the SI ATRP method. % CBMAA (poly (HPMAA-coCBMAA / 15 mol%) with a thickness in the range of 19 to 31 nm according to the protocol described in PV 2015313. Subsequently, the chip was rinsed with water and mounted in the SPR sensor chamber with 6 flow microfluidic channels. in channels 2 to 6 activated by reaction with aqueous solution of Λ-hydroxysuccinimide (NHS; 0.1 M) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; 0.5 M) for 20 min at 2 ° C ( step (a) in Example 1) After rinsing with water for 5 min and PBS and water pH 8 (10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , 137 mM NaCl, 2.7 mM KCl, pH 7, 4) The activated brush was exposed to glycine solution (1 M, water pH 8) for 30 min in the 3rd channel, (2-aminoethoxy) acetic acid solution (0.1 M, water pH 8) for 30 min in the 4th channel , ethanolamine solution (1 M, water pH 8) for 30 min in channel 5 and deactivation buffer (10 mM sodium borate + 10 mM imidazole + 10 mM NaCl, pH 8) 40 min in channel 6 at 20 ° C (step (c) v Example 1) raven SPR sensor in which the first channel contained

-6GB 307026 B6 Non-activated brush, 2nd channel brush after activation, 3rd channel brush after deactivation with glycine, 4th channel brush after deactivation with (2-aminoethoxy) acetic acid, 5th channel brush after deactivation with ethanolamine and 6th channel brush after deactivation with deactivation buffer. Each time the brush was modified in the appropriate channel, the brush was rinsed with water for 5 min and PBS for 10 min, SPR resonance wavelength (λ) was measured. The surface was incubated with undiluted blood plasma for 10 min, rinsed with PBS and again measured λ ₂ . The amount of biological deposit in ng / cm ² was calculated from the difference λ ₂ -λ].

In the same way, biological deposits were measured on other chips from complex food samples prepared by homogenization according to the protocol described in ČSN ISO 7251 and ČSN EN ISO 6579. The results are shown in Tables 1 and 2.

Table 1: Deposition from complex biological solutions onto poly (HPMAA-coCBMAA / 15 mol%) brush activated with EDC / NHS and inactivated with deactivating agents.

Deactivating agent Deposit [ng / cm] Blood plasma Milk Hamburger Cucumber Before activation 4.9 ± 2.0 3.1 0.0 0.0 After activation 135.5 74.3 55.3 21.4 Glycine ' ¹ 5.1 1.9 1.4 0.2 (2-Aminoethoxy) acetic acid ² 5.9 1.9 0.7 1.4 Ethanolamine 14.8 11.4 6.6 1.4 Deactivation buffer ⁴ 12.5 ± 3.1 5.8 2.4 3.4

* '1 M glycine, pH 8, 30 min * ² Loom acid (2-aminoethoxy) acetic acid, pH 5, 30 min * ³ 1 M ethanolamine, pH 8, 30 min * ⁴ mM sodium borate + mM imidazole + mM NaCl, pH 8 , 40 min

Table 2 Deposition from complex biological solutions onto polyCBMAA activated brush

EDC / NHS and deactivated with deactivating agents

Deactivating agent 2 Deposit [ng / cm] Blood plasma Milk Hamburger Cucumber Before activation 9.6 ± 1.3 10.7 4.1 10.9 Immediately after activation 222.7 897.6 73.1 135.5 Glycine ' ¹ 75.7 317.9 11.2 13.6 (2-Aminoethoxy) acetic acid 71.9 316.1 14.6 11.1 Ethanolamine ' ³ 90.4 364.6 16.3 15.1 Deactivation buffer ⁴ 92.9 380.8 63.9 43.4

* '1 M acetic acid * ² Loom (2-aminoethoxy) acetic acid, pH 5, 30 min * ³ 1 M ethanolamine, pH 8, 30 min * ⁴ mM sodium borate mM imidazole + + mM NaCl, pH 8, 40 min

Tables 1 and 2 show significantly reduced deposition from all biological samples tested on both types of carboxybetain polymer brushes after inactivation by covalent coupling of amino acid reagents (glycine or NH ₂ -CH ₂ -CH ₂ -O-CH ₂ -COOH) compared to the inactivated inactivation by hydrolysis in the deactivation buffer or by ethanolamine binding.

Claims (9)

PATENT CLAIMS A method of preparing a surface of a substrate containing carboxybetaine groups, with increased resistance to undesirable deposition of biological media components on the substrate surface, comprising the steps of: (a) chemical activation of carboxybetain groups on the surface of the substrate by converting the carboxyl group of the carboxybetain to the active ester; b) covalently attaching the bioactive agent by reacting its amino group with the active ester prepared in step a), characterized in that after step b) step c) is carried out in which the product of step b) is reacted with (2-aminoethoxy) acetic acid, active esters are reacted with (2-aminoethoxy) acetic acid and the bioactive agent is not covalently bound in step b). Method according to claim 1, characterized in that step c) is carried out by first washing the product of step b) with the buffer used as solvent in step b), then 307026 B6 is rinsed with the buffer that will be used in the next incubation step, then the product is incubated with a solution of (2-aminoethoxy) acetic acid in the buffer, followed by rinsing with the buffer used for incubation, followed by rinsing with the solution loaded for storage or with water and dried. Method according to any one of the preceding claims, characterized in that the bioactive agent is a substance comprising at least one NH ₂ group and selectively interacting with a target component of the biological medium, preferably the bioactive agent is a substance with affinity for the target component selected from the group comprising an antibody, antigen, lectin, cell receptor and analogs thereof, recombinantly prepared portions, synthetic oligopeptide sequences, nucleic acids and portions thereof, synthetic oligonucleotide sequences, aptamers; a chemical catalysing agent for a target substance selected from the group consisting of enzymes, coenzymes, and synthetic analogs thereof; a biological response agent selected from the group consisting of anticoagulants, proteins and oligopeptide sequences responsive to cellular integrins, growth factors, hormones, and drugs. Method according to any one of the preceding claims, characterized in that the biological medium is a fluid containing biomolecules and their associates selected from the group consisting of proteins, carbohydrates, polysaccharides, lipids, nucleic acids, lipoproteins, glycoproteins, organelles; cells, viruses and microorganisms and / or fragments thereof. Method according to any one of the preceding claims, characterized in that the carboxybetaine groups are contained in a polymer layer on the surface of the substrate selected from the group comprising a polymer brush prepared by polymerizing or grafting polymers onto the surface of the substrate, and preferably having a polymer coating. this layer has a thickness ranging from 5 nm to 5 µm. 6. The process of claim 5 wherein the polymer layer comprises at least one polymer containing carboxybetaine groups. The method of claim 5, wherein the polymer layer containing carboxybetaine groups is a polymer brush of poly (carboxybetaine methacrylamide), poly (carboxybetaine methacrylate), poly (carboxybetaine acrylamide) or poly (A-co-B) copolymer. wherein A is a monomeric unit of the group N - (2-hydroxypropyl) methacrylamide, 2-hydroxyethyl methacrylate, 3-hydroxypropyl methacrylate and oligo (hydroxyethylene glycol) methacrylate and B is a monomeric unit of the group carboxybetain methacrylamide, carboxybetain methacrylate and carboxybetain concentration from 1 to 99 mol%. Method according to any one of the preceding claims, characterized in that the substrate is selected from the group consisting of particles preferably in a size range of 5 nm to 1 mm, porous membranes, cell carriers and biosensors. The method of claim 8, wherein the substrate is a biosensor detection surface for direct or multi-step detection of analytes in complex biological media.